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Thiamine transporter-2 deficiency is caused by mutations in the SLC19A3 gene. As opposed to other causes of Leigh syndrome, early administration of thiamine and biotin has a dramatic and immediate clinical effect. New biochemical markers are needed to aid in early diagnosis and timely therapeutic intervention. Thiamine derivatives were analysed by high performance liquid chromatography in 106 whole blood and 38 cerebrospinal fluid samples from paediatric controls, 16 cerebrospinal fluid samples from patients with Leigh syndrome, six of whom harboured mutations in the SLC19A3 gene, and 49 patients with other neurological disorders. Free-thiamine was remarkably reduced in the cerebrospinal fluid of five SLC19A3 patients before treatment. In contrast, free-thiamine was slightly decreased in 15.2% of patients with other neurological conditions, and above the reference range in one SLC19A3 patient on thiamine supplementation. We also observed a severe deficiency of free-thiamine and low levels of thiamine diphosphate in fibroblasts from SLC19A3 patients. Surprisingly, pyruvate dehydrogenase activity and mitochondrial substrate oxidation rates were within the control range. Thiamine derivatives normalized after the addition of thiamine to the culture medium. In conclusion, we found a profound deficiency of free-thiamine in the CSF and fibroblasts of patients with thiamine transporter-2 deficiency. Thiamine supplementation led to clinical improvement in patients early treated and restored thiamine values in fibroblasts and cerebrospinal fluid.

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Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient's fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.

We conducted a literature review of all reported cases with these genetic defects, and focused our paper on treatment efficacy and safety, adverse effects, dosing and treatment monitoring. Expert commentary: Doses of thiamine vary according to the genetic defect: for SLC19A2, the usual dose is 25-200 mg/day (1-4 mg/kg per day), for SLC19A3, 10-40 mg/kg per day, and for TPK1, 30 mg/kg per day. Thiamine supplementation in SLC19A3-mutated patients restores CSF and intracellular thiamine levels, resulting in successful clinical benefits. In conclusion, evidence collected so far suggests that the administration of thiamine improves outcome in SLC19A-2, SLC19A3- and TPK1-mutated patients, so most efforts should be aimed at early diagnosis of these disorders.

This article is available online at http://www.jlr.org Niemann-Pick type C (NPC; MIM#257220) is a progressive neurodegenerative disease caused by a disorder in the intracellular traffi cking of cholesterol that leads to a lysosomal/endosomal accumulation of unesterifi ed cholesterol and glycolipids in many tissues ( 1-3 ). The detection of free cholesterol accumulation by fi lipin staining in fibroblasts has been for many years the gold standard methodology for the biochemical diagnosis of the disease. This method has good sensitivity and specifi city. However, juvenile or adult onset forms sometimes present interpretation diffi culties ( 4 ). In addition, the method involves an invasive skin biopsy and the culture of fi broblasts that requires several weeks for the cellular growth, which delays the diagnosis. Some studies in cellular and animal models showed a correlation between lipid accumulation and cellular oxidative stress that produces an increase of reactive oxygen species and lipid peroxidation ( 5-8 ). In the NPC murine model, an increase of two oxysterols, cholestane-3 b ,5 a ,6 b -triol (CT) and 7-ketocholesterol (7-KC), has been observed ( 8 ). This evidence was later confi rmed in NPC patients (8)(9)(10)(11)(12)(13), showing a correlation between the oxysterol profi le and the age of onset and severity of the disease ( 8,14 ). Moreover, CT specifi cally has been found to be increased in NPC disease compared with other lysosomal and neurodegenerative diseases ( 8 ). These results Abstract Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterifi ed cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3 ␤ ,5 ␣ ,6 ␤ -triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specifi city of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 pa- 26,26,26,27,27, ; AUC, area under receiver-operating characteristic curve; BHT, butylhydroxytoluene; CI, confi dence interval; CT, cholestane-3 b ,5 a ,6 b -triol; 5 a ,26,26,26,27,27, ; CTX, cerebrotendinous xanthomatosis; % CV, percentage coeffi cient of variation; LAL, lysosomal acid lipase; LOD, limit of detection; LOQ, limit of quantifi cation; LSD, lysosomal storage disease; MRM, multiple reaction monitoring; NPC, Niemann-Pick type C; ROC, receiver-operating characteristic; SLO, Smith-Lemli-Opitz.

We describe the first two unrelated Spanish patients with creatine transporter deficiency initially identified by brain proton magnetic resonance spectroscopy (MRS). The clinical phenotype was characterized by severe mental retardation, epilepsy, autism, severe speech delay and absence of brain creatine by MRS. Urine creatine/creatinine ratio was increased and creatine uptake in fibroblasts was impaired in both patients. On DNA sequence analysis of the SLC6A8/creatine transporter gene, one hemizygous mutation was found in each patient: one mutation was novel and consisted of a deletion of two nucleotides c.878-879delTC in exon 5, resulting in a frameshift (p.Lys293fsX3), and in the other patient a known deletion of three nucleotides 1222-1224delTTC in exon 8 resulting in p.Phe408del. Creatine treatment for one year failed to improve the neurological symptoms and was associated with a striking increase in body weight in both patients (13 and 16 kg, respectively).